stst uctu eructure-functionunction elationship relationship
TRANSCRIPT
Structure-Function RelationshipSt uctu e unction elationship
Bhimu PatilDirector, Vegetable and Fruit Improvement Center, g p
Professor, Department of Horticultural SciencesTexas A&M UniversityTexas A&M University
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
Structure-Activity Relationship (SAR) is a means by which the effect of a drug or bioactive moleculeby which the effect of a drug or bioactive molecule on animal, plant, or the environment can be related
to its molecular structureto its molecular structureor
The relationship between the chemical structure ofThe relationship between the chemical structure of a compound and its biological or pharmacological
activityy
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
F i fl i SARFactors influencing SAR
• What are the properties that describe a molecule?molecule?
• Molecular structureSh• Shape
• Size• Stereochemical arrangements
S iStereoisomers
Isomers which have their atoms connected in the same sequence but differ in the way the atoms are oriented in space - i.e. the difference between two stereoisomer lies only inbetween two stereoisomer lies only in the three dimensional arrangement of atoms. Stereoisomers may beof atoms. Stereoisomers may be classed as cis-trans isomers or optical isomers
Recent scientific understanding taste ecent scientific unde standing taste
Chandrashekar et al., 2006. Nature:444:288-294
Stereochemistry
+L‐aspartic acid has a flat taste L‐phenylalanine tastes bitter
L‐Aspartame L‐Aspartame
I am
I am bitter!
D‐Aspartame
sweet!
L‐Aspartame :Taste‐200 times more sweeter than sucroseD‐Aspartame: taste bitter
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
Li id Vit CLimonoids Vit. C Flavonoids
CarotenoidsPotassiumotass u
PectinFiber
F li A idCoumarins Folic AcidCoumarins
C L dCitrus Limonoids
• 54 known limonoids (Triterpenoids)O22
– Present as
O
O
3 12 1718 20
21 23
• Aglycones • Glucosides
O
OO
O
O
A
A'
B
CD
1
2
5 78
910
11 1314
15
161924
GlucosidesO
Limonin
4
25a25b
5
6
7
Structure of Limonin
1 Li i 19 6 Keto 7β deacet lnomilol
LIMONOIDS AGLYCONES1. Limonin
2. Nomilin
3. Obacunone
4 Deacetylnomilin
19. 6-Keto-7β-deacetylnomilol
20. 6-Keto-7 β-Nominol
21. Isocylocalamin
22. 1-(10-19)Abeo-7 α-acetoxy-10 β-4. Deacetylnomilin
5. Ichangin
6. Deoxylimonin
7 Deoxylimonol
( ) y βhydroxyisoobacunoic acid 3,10-lactone
23. 1-(10-19)Abeo-obacun-9(11)-en-7 α0yl acetate
24. Deacetylnomilinic acid7. Deoxylimonol
8. Limonol
9. Limonyl acetate
10 7α-Obacunyl acetate
24. Deacetylnomilinic acid
25. Nomilinic acid
26. Iso-Obacunoic acid
27. Deoxylimonic acid10. 7α-Obacunyl acetate
11. 7α-Obacunol
12. Ichangensin
13. Citusin
28. Isolimonic acid
29. Epi-iso-obacunoic acid
30. 17-Dehydrolimonoic acid A ring lactone
31 Calaminic acid13. Citusin
14. Calamin
15. Retrocalamin
16. Cyclocalamin
31. Calaminic acid
32. Retrocalaminic acid
33. Cyclocalaminic acid
34. Iso-Obacunoic acid disophenol16. Cyclocalamin
17. Methyl-iso-obacunoate disosphenol
18. Methyl deacetylnomilinate
p
35. Obacunoic acid
36. 19-Hydroxydeacetylnomilinic acid
37. Limonexic acid
Monocarboxylic acids
17-β-D-Glucopyranosides of:
Dicarboxylic acids
17-β-D-Glucopyranosides of:
1. Limonin
2 N ili
10. Nomilinic acid
11 D t l ili i id2. Nomilin
3. Obacunone
4. Ichangensin
11. Deacetylnomilinic acid
12. Obacunoic acid
13. Trans-Obacunoic acidg
5. Deacetylnomilin
6. Ichangin
14. Iso-Obacunoic acid
15. Epi-iso-obacunoic acid
7. Calamin
8. Methyl deacetyl nomilinate
16. Isolimonic acid
17. 19-Hydroxydeacetyl nomilinic acid
9. 6-Keto-7β-deacetylnomilol
Major limonoid found in citrusMajor limonoid found in citrus
Hb = the total maximal number of hydrogen bonds possible either as a H-donor / acceptor.Clogp = calculated octanol/water partition coefficient on the logarithmic scale.
Antifeedant activity of limonoidsLimonin and ten structurally modified limonins were evaluated as antifeedants against 4th instar larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say
Li iLimonin
O
OO
O
O
O
O
O
OH
OO
O
Epilimonol
O
O
OO
O
O
O
O
OH
OO
O
O
Results: The epoxide and furan groups were shown to be essential structural requirements for high antifeedant activity
TetrahydrolimoninDeoxylimoninDeoxylimonol
requirements for high antifeedant activity
Bentley et al., 1988. Entomologia Experimentalis et Applicata. 49: 189-193.
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
Phase I Enzymes
C
Pro
CYP’s DNA DAMAGE
ProCarcinogens
Ultimate Phase II Enzyme Carcinogen
GST, QR, UDPGTEXCRETION
+GSH
, Q ,
Conjugated product
H
Conjugated product
(adapted and modified from Wilkinson & Clapper 1997)
Wh h II ?What are phase –II enzymes?
• A group of xenobiotic metabolizing enzymes that are mainly involved in the inactivation and
i f i d h iexcretion of carcinogens and other toxic chemical substances.Hence compounds known to induce phase IIHence compounds known to induce phase-II enzymes are considered to be potential cancer preventive.
Ex: GST( Glutathione S-transferase), QR (Quinone Reductase), UDP-glucuronosyltransferase (UGT), and sulfotransferase (ST)sulfotransferase (ST)
Structure of purified and modified citrus limonoidscitrus limonoids
Perez et al J Agric Food Chem 2009 57 5279 5286Perez et al., J. Agric. Food Chem. 2009, 57, 5279-5286.
GST Activity against 1-Chloro-2,4-dinitrobenzene
Sample stomach intestine liver lungcontrol 0.88 ± 0.07 0.86 ± 0.09 1.78 ± 0.02 0.36 ± 0.08
GST Activity against 1 Chloro 2,4 dinitrobenzene
limonin 0.83 ± 0.13 0.63 ± 0.01 1.63 ± 0.27 0.34 ± 0.05limonin-7-methoxime 0.89 ± 0.18 1.34 ± 0.47 2.36 ± 0.05** 0.36 ± 0.03
d f li i 0 86 0 12 0 79 0 09 1 45 0 54 0 34 0 08defuran limonin 0.86 ± 0.12 0.79 ± 0.09 1.45 ± 0.54 0.34 ± 0.08LG 0.86 ± 0.13 0.75 ± 0.06 1.41 ± 0.08* 0.44 ± 0.09DNAG 1.15 ± 0.38 0.70 ± 0.13 1.35 ± 0.20* 0.60 ± 0.12**
Specific activity (units/mg protein). The results are the means ± SD (n = 4). *indicates statistically significant (p < 0.05) decrease of activity using Student’s t-test. ** indicates statistically significant (p < 0.05) induction of activity using Student’s t-test
Perez et al., J. Agric. Food Chem. 2009, 57, 5279-5286.
LimoninLimonin 7- methoxime
GST activity in different organ homogenates against 4-nitroquinoline 1-oxide, a potent xenobiotic tumorigenic to the lung, esophagus, forestomach, glandular stomach, skin, and other organs. g , , g
The bars indicate the mean ± SD (n = 4). * indicates statistically significant (p < 0.05) decrease in activity. ** indicates statistically significant (p < 0.05) induction using Student’s t-test.
Perez et al., J. Agric. Food Chem. 2009, 57, 5279-5286.
Quinone Reductase ActivityQuinone Reductase ActivitySAMPLE STOMACH INTESTINE LIVER LUNG
control 8.30 ± 1.73 2.33 ± 0.27 0.52 ± 0.04 0.26 ± 0.05control 8.30 ± 1.73 2.33 ± 0.27 0.52 ± 0.04 0.26 ± 0.05
limonin 8.56 ± 1.58 2.47 ± 0.72 0.38 ± 0.10 0.29 ± 0.04
limonin-7-methoxime
10.45 ± 1.12 4.01 ± 1.15 0.85 ± 0.11** 0.34 ± 0.01**
defuran limonin 8.98 ± 0.79 2.00 ± 0.24 0.41 ± 0.02 0.38 ± 0.05**
LG 8.12 ± 2.38 2.45 ± 0.14 0.47 ± 0.06 0.31 ± 0.05
DNAG 9.58 ± 4.07 2.29 ± 0.50 0.23 ± 0.02* 0.28 ± 0.01
Specific activity (units/mg protein). The results are the means ± SD (n = 4). * indicates p y ( g p ) ( )statistically significant (p < 0.05) decrease of activity using Student’s t-test. ** indicates statistically significant (p < 0.05) induction of activity using Student’s t-test.
Perez et al., J. Agric. Food Chem. 2009, 57, 5279-5286.
CurcuminPolyphenolic compound
Chemically DiferuloylmethaneC e ca y e u oy et a e
Rhizomes of Turmeric (Curcuma longa) perennial plant of the ginger familyplant of the ginger family
History of Turmeric• Ayurvedia: Ancient system of health care that is native to
the Indian subcontinent (3000 BC)
• “I have found a plant that has all the qualities of Saffron• I have found a plant that has all the qualities of Saffron, but it is a root” (Marco Polo on Turmeric, 1280 AD)
• Used in India at least since 6000 years
• Europe rediscovered it 700 years ago via Marco Polo and it is used in traditional Brazilian medicine
RECENT CLINICAL STUDIESDose (g/day) Subjects Results References
0.5 – 12g daily,up to 6 months
Patients with pre-neoplasia (25)
Delay of progression – full blown malignancy
Cheng, Anticancer Res. 2001, 21, 2895 –2900.(Taiwan)
2 daily, 6 months/ sulfasalazine
Patients - ulcerative colitis (43)
Prevent relapse Holt, et al., Digest Dis. Sci. 2005, 50, 2191
0.45– 3.6g. daily7 days
Patients with colorectal cancer (23)
Reduction in Serum levels of prostaglandin E-2
Garcea, G. Cancer Epidemiol. BiomarkersPrev. 2005, 14, 120–125.
0.5-12 g / single Healthy volunteers (24) No –toxicity Lao et al., BMC, Complement. Alternate. Med. 2006, 6, 10.
1.1 – 1.65 daily, - Patients -ulcerative Delay in disease Hanai, H., Clin. G t t l H t l
y,3 months colitis/ Crohn’s
disease(10)
yprogression
Gastroenterol. Hepatol. 2006, 4, 1502 –1506.
Structure Function relationship of Curcumin to induce Phase-II enzyme
The study was conduced to understand the S i iStructural requirement to induce Phases-II enzyme (Quinone Reductase) using in murine hepatoma cells.
Structures of curcuminoids tested in this study and their inducer potencies (CD values) in the QR assay in Hepa1c1c7 murine
hepatoma cells
Dinkova-Kostova, A. T. et al. Carcinogenesis 1999 20:911-914; 1
Wh h d l d ?What this study concludes?The presence of two structural elements was found to
be required for high inducer potency:• (i) hydroxyl groups at ortho-position on the
aromatic rings andaromatic rings and• (ii) The β-diketone
LOW TEMP / PH
Diketo form Keto-enol form
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
QUORUM SENSINGHow
much is youryour
count?
Q i i d fi d d i d dQuorum sensing is defined as density dependent regulation of gene expression at population l l Th h i i lllevel. The phenomenon was originally identified in marine bacterium Vibrio fisheri.
QUORUM SENSINGQUORUM SENSING
• Cell-cell communication• Multi cellular behavior• Multi-cellular behavior• Signaling molecules
Si li M l lSignaling Molecules
• N-Acylhomoserine Lactones (AHL)• Furanosyl borate dieaster (Autoinducer-2)• Small peptides• Autoinducer-3 (structure unknown)
B
OHHO
O O
HO
OHO
Furanosyl borate diester
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
Modified Analogues of Limonoids
O
OOO
OHOO
O
tura
l
d
O
OO
O
O
O
A B
C DA'
O
O
O
OO
O
O
O
OO
O
O GluNa
Mod
ified
O LimoninB
D-furan Limonin Limonin Glucoside
O OModified
O
O
O
OO
OO
O
O
OO
O
O O
NOH
Limonin-7-Oxime
NO CH3
Limonin-7-Methoxime
Antagonistic activity towards cell-cell g ysignaling by Limonin-7-Oxime
Inhibition of AI-1 mediated Cell Signaling by Limonin and Limonin-7-Oxime
506070
on
LimoninLimonin-7-Oxime
Inhibition of AI-2 mediated Cell Signaling by Limonin and Limonin-7-Oxime
60
80
on
01020304050
6.25 12.5 25 50 100
Concentration (μg/ml)
% In
hibi
tio
0
20
40
6.25 12.5 25 50 100Concentration (μg/ml)
% In
hibi
tio
Limonin Limonin-7-Oxime(μg )
Inhibition of Biofilm formation by Limonin and Limonin-7-Oxime
60LimoninLimonin-7-Oxime O
O
O
01020304050
6.25 12.5 25 50 100
% In
hibi
tion
Limonin 7 OximeO
O
O
OO
NOH
Concentration (μg/ml)OH
Limonin-7-Oxime
Antagonistic activity towards cell-cellAntagonistic activity towards cell cell signaling by Limonin-7-Methoxime
Inhibition of AI-1 mediated Cell Signaling by Limonin and Limonin-7-Methoxime
40506070
hibi
tion
LimoninLimonin-7-Methoxime
Inhibition of AI-2 mediated Cell Signaling by Limonin and Limonin-7-Methoxime
40
60
80
ibiti
on
LimoninLimonin-7-Methoxime
0102030
6.25 12.5 25 50 100
Concentration (μg/ml)
% In
h
0
20
40
6.25 12.5 25 50 100
Concentration (μg/ml)
% In
h
Inhibition of Biofilm formation by Limonin and Limonin-7-Methoxime
5060
n
LimoninLimonin-7-Methoxime OO
O
O
01020304050
6.25 12.5 25 50 100
% In
hibi
tion
OOO
NO CH3
Li i 7 M th iConcentration (μg/ml) Limonin-7-Methoxime
Antagonistic activity towards cell-cell g ysignaling by D-furan limonin
Inhibition of AI-1 mediated Cell Signaling by Limonin and D-Furan Limonin
6070
n
LimoninD-Furan Limonin
Inhibition of AI-2 mediated Cell Signaling by Limonin and D-Furan Limonin
60
80
n
01020304050
6.25 12.5 25 50 100
C t ti ( / l)
% In
hibi
tion D Furan Limonin
0
20
40
60
6.25 12.5 25 50 100
C t ti ( / l)
% In
hibi
tion
LimoninConcentration (μg/ml) Concentration (μg/ml)D-Furan Limonin
Inhibition of Biofilm formation by Limonin and D-Furan Limonin
40LimoninD-Furan Limonin OO
OOHO
0
10
20
30
6.25 12.5 25 50 100
% In
hibi
tion
OOO
O
D f Li iConcentration (μg/ml)
D-furan Limonin
Moderate difference in antagonistic activity ff g ytowards cell-cell signaling by Limonin Glucoside
Inhibition of AI-1 mediated Cell Signaling by Limonin and Limonin Glucoside
40506070
ibiti
on
LimoninLimonin Glucoside
Inhibition of AI-2 mediated Cell Signaling by Limonin and Limonin Glucoside
60
80
100
bitio
n
0102030
6.25 12.5 25 50 100
Concentration (μg/ml)
% In
hi
0
20
40
6.25 12.5 25 50 100
Concentration (μg/ml)
% In
hi
LimoninLimonin Glucoside
Inhibition of Biofilm formation by Limonin and Limonin Glucoside
40 LimoninLimonin Glucoside
O
O
0
10
20
30
6.25 12.5 25 50 100
% In
hibi
tion
Limonin GlucosideO
O
OO
O
O Glu
Concentration (μg/ml)Limonin Glucoside
• Modification of 7th position reduces the potency in bioluminescence assays
• However, the same modifications enhanced the potency in biofilm assayspotency in biofilm assays
• Addition of glucose molecule at 17th position reduces g pthe moderate potency in bioluminescence assays as well as in biofilm assays
• Furan ring and 7th position are critical for antagonistic activity against cell-cellfor antagonistic activity against cell cell signaling
• However• However…– Seventh position is important for AI-1 activity
inhibition– Furan ring is probably critical for AI-2 activity
inhibition
B f lBiofilm
• Polysaccharide matrix• Different bacterial cell physiology• Different bacterial cell physiology• Cause recurrence of infection• Resistant/tolerant• Notorious
Effect of Limonin -7-methoxime on ff fdifferent bacterial adhesins
40
50
10
20
30
hibi
tion
-10
0
10
6.25 12.5 25 50 100
% In
h
-20
-10
Concentration (μg/ml)(μg )Pilli Inducing Ag43 Inducing Curli Inducing Curli Non-inducing
Effect of Limonin -7-oxime on different bacterial adhesins
40
2030
40
on
010
6 25 12 5 25 50 100
Inhi
bitio
-30
-20-10 6.25 12.5 25 50 100%
30
Concentration (μg/ml)
Pilli Inducing Ag43 Inducing Curli Inducing Curli Non-inducingPilli Inducing Ag43 Inducing Curli Inducing Curli Non-inducing
C l iConclusion
• Modified limonoids Limonin 7-oxime and limonin 7-methoxime inhibit biofilm by alimonin 7 methoxime inhibit biofilm by a different mechanism.
O liOutline• Definition• Definition• Descriptors• Limonoids Antifeedant Activity• Limonoids-Antifeedant Activity• Phase II Enzymes inducers
Limonoids– Limonoids– Curcumin
• Quorum Sensing• Quorum Sensing– Limonoids– FlavonoidsFlavonoids
Presence of double bond between 2-3 fposition (Apigenin)
OH
OHOOH
OH O
OHO
Naringenin
OH O
A i iApigenin
Reduction in antagonistic activity towards cell-g ycell signaling and biofilm formation.
Inhibition of AI-2 mediated cell-cell signaling by Naringenin and Apigenin
100.00
0.00
20.00
40.00
60.00
80.00
% In
hibi
tion
6.25 12.5 25 50 100
Concentration (μg/ml)Naringenin ApigeninInhibition of biofilm formation by Naringenin and
Apigenin
75
on
0
25
50
6 25 12 5 25 50 100
% In
hibi
tio
6.25 12.5 25 50 100
Concentraton (μg/ml)Naringenin Apigenin
Addition of hydroxyl group at positionAddition of hydroxyl group at position 3 (Kaempferol)
OH
OHOHO
OHOOH O
Apigenin
OH O
OHApigenin
Kaempferol
Increase in antagonistic activity towards cell-cell signaling and biofilm formation.
Inhibition of AI-2 mediated cell-cell signaling by Apigenein and Kaempferol
80
100
0
20
40
60
80
% In
hibi
tion
6.25 12.5 25 50 100Concentration (μg/ml)Apigenin Kaempferol
Inhibition of Biofilm formation by Apigenin and Kaempferol
40.00n
0.00
10.00
20.00
30.00
% In
hibi
tion
6.25 12.5 25 50 100
Concentration (μg/ml)Apigenin Kaempferol
Addition of hydroxyl group at position 5’ (Quercetin)
OHO
OH
OHO
OHOH
OH O
OH
Kaempferol
OHOOH
Kaempferol
OH O
OH
Quercetin
I i t i ti ti it t d llIncrease in antagonistic activity towards cell-cell signaling and biofilm formation.
Inhibition of AI-2 mediated cell-cell signaling by Kaempferol and Quercetin
95
100
n
80
85
90
95
% In
hibi
tion
6.25 12.5 25 50 100
Concentration (μg/ml)Kaempferol QuercetinInhibition of Biofilm formation by Kaempferol and
Quercetin
80.00
20.00
40.00
60.00
80.00%
Inhi
bitio
n
0.006.25 12.5 25 50 100
Concentration (μg/ml)Kaempferol Quercetin
Addition of rutinoside at position 3 ddition of utinoside at position 3(Rutin)
OHOH
OHOOH
OHO
OH
OH
OH O
O
OO
OH
OH O
Quercetin
OH
O
OH
OHOHOHOH
Rutin
Reduction in antagonistic activity towards cell-g ycell signaling and biofilm formation.
Inhibition of AI-2 mediated cell-cell signaling by Quercetin and Rutin
80.00
100.00
n
0.00
20.00
40.00
60.00
80.00
% In
hibi
tion
6.25 12.5 25 50 10
Concentration ( μg/ml)Quercetin Rutin Inhibition of Biofilm formation by Quercetin and Rutin
7080
0102030405060
% In
hibi
tion
06.25 12.5 25 50 100
Concentration (μg/ml)Quercetin Rutin
C l iConclusion
Presence of double bond between position 2 and 3 is detrimentalAddition of hydroxyl groups increases the potencyPosition 3 is important for the activity against AI-2 and biofilm 3'against AI 2 and biofilm
O
A
B
C
127
8 1'
2' 4'
5'
6'
3
45
6